U.S. patent application number 14/544755 was filed with the patent office on 2015-10-22 for apparatus for enhanced recovery of regenerative cells from tissue samples.
This patent application is currently assigned to InGeneron Incorporated. The applicant listed for this patent is InGeneron Incorporated. Invention is credited to Eckhard U. Alt, Michael E. Coleman, Ron Stubbers.
Application Number | 20150299643 14/544755 |
Document ID | / |
Family ID | 46245409 |
Filed Date | 2015-10-22 |
United States Patent
Application |
20150299643 |
Kind Code |
A1 |
Alt; Eckhard U. ; et
al. |
October 22, 2015 |
Apparatus for enhanced recovery of regenerative cells from tissue
samples
Abstract
This document describes methods and an apparatus for recovery of
a cell-enriched matrix and cells (e.g., regenerative cells) from a
tissue sample. In some embodiments, at least two rounds of
acceleration and deceleration are performed.
Inventors: |
Alt; Eckhard U.; (Houston,
TX) ; Stubbers; Ron; (Houston, TX) ; Coleman;
Michael E.; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
InGeneron Incorporated |
Houston |
TX |
US |
|
|
Assignee: |
InGeneron Incorporated
Houston
TX
|
Family ID: |
46245409 |
Appl. No.: |
14/544755 |
Filed: |
February 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13998079 |
Sep 27, 2013 |
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14544755 |
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13329143 |
Dec 16, 2011 |
8951513 |
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13998079 |
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13385599 |
Nov 3, 2011 |
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13329143 |
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61424012 |
Dec 16, 2010 |
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Current U.S.
Class: |
435/307.1 ;
435/283.1 |
Current CPC
Class: |
A01N 1/0242 20130101;
C12N 2509/10 20130101; C12N 5/0653 20130101; A61L 2430/34 20130101;
C12M 47/02 20130101; C12M 41/12 20130101; B04B 15/02 20130101; C12N
2509/00 20130101; C12M 47/04 20130101; A61L 27/3604 20130101; A61P
43/00 20180101; A61K 35/35 20130101; B04B 5/0414 20130101; C12N
5/0667 20130101; C12M 33/10 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12M 1/26 20060101 C12M001/26; A01N 1/02 20060101
A01N001/02; C12M 1/34 20060101 C12M001/34 |
Claims
1. A apparatus for processing samples of body tissue having
vasculature and having regenerative cells adaptive to differentiate
into functional equivalent of host tissue after transplantation
therein, the apparatus comprising an orifice for extruding a sample
of the body tissue therethrough to produce an extruded tissue
sample having reduced lipid content and increased regenerative cell
content relative to the lipid content and resident regenerative
cell content therein prior to the extrusion; and a centrifuge for
receiving and subjecting the extruded tissue sample to
centrifugation to further increase regenerative cell content
therein, whereby to produce a cell-enriched matrix therefrom.
2. The apparatus of claim 1, wherein body tissue samples to be
processed are adipose, lipoaspirate, or a combination thereof.
3. The apparatus of claim 1, further comprising a second centrifuge
for receiving and subjecting the cell-enriched matrix to repetitive
rounds of rotational acceleration and deceleration in an enzymatic
environment to dissociate and extract regenerative cells from the
cell-enriched matrix, for combination of the extracted regenerative
cells with other cell-enriched matrix into a cellular composition
having enhanced neovascularization.
4. The apparatus of claim 1, wherein the body tissue samples to be
processed are obtained directly from a living subject or from a
tissue bank in which the samples have been cryopreserved and
stored.
5. The apparatus of claim 3, including a closed transfer system the
first-named and additional centrifuges to enable transfer of the
cell-enriched matrix from the first-named centrifuge to the second
centrifuge.
6. The apparatus of claim 2, wherein adipose body tissue samples
are initially processed into suspension of pieces of adipose tissue
in aqueous solution, prior to extrusion.
7. The apparatus of claim 1, wherein the orifice has a diameter in
a range from about 1 mm to about 5 mm.
8. The apparatus of claim 3 including a controller for maintaining
a selected temperature in a range from about 26.degree. C. to about
42.degree. C. interiorly of said second centrifuge during
centrifugation of cell-enriched matrix therein to increase the
speed of enzymatic dissociation of the regenerative cells.
9. The apparatus of claim 1, wherein the extruded tissue sample is
subjected to centrifugation for at least about five minutes at a
force in a range from about 400 g to about 1200 g.
10. (canceled)
11. The apparatus of claim 3, wherein said repetitive rounds of
rotational acceleration and deceleration are substantially
uninterrupted.
12. The apparatus of claim 3, further comprising a cryopreserver
arranged and adapted to process the cellular composition for
banking.
13. The apparatus of claim 3, further comprising a collector for
introducing the extracted regenerative cells into a resorbable
scaffold.
14. The apparatus of claim 13, further comprising a cryopreserver
arranged and adapted to process the resorbable scaffold and
regenerative cells therein for banking.
15. The apparatus of claim 13, further comprising a bank for
storing said resorbable scaffold and regenerative cells therein in
a preserved state.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Nonprovisional
application Ser. No. 13/998,079, filed Sep. 27, 2013, which is a
division of U.S. Nonprovisional application Ser. No. 13/329,143,
filed Dec. 16, 2011, now U.S. Pat. No. 8,951,513, issued on even
date herewith, which claims priority to U.S. Provisional
Application No. 61/424,012, filed Dec. 16, 2010, now expired, and
to U.S. Provisional Application No. 61/555,305, filed Nov. 3, 2011,
which was converted to U.S. Nonprovisional application Ser. No.
13/385,599, now abandoned, and is a continuation-in-part of both
the '012 provisional application and the '599 nonprovisional
application. The disclosures of the prior applications are hereby
incorporated in their entireties by reference in the disclosure of
this application. Further, this application is related to U.S.
Nonprovisional applications designated by Attorney Docket Nos.
30254.2745DIV2 and 30254.2745DIV3, both of which were filed on even
date herewith.
TECHNICAL FIELD
[0002] This invention relates to methods and an apparatus for
recovery of a cell-enriched matrix and recovery of cells, and more
particularly to recovery of cells from a tissue sample using two or
more acceleration and deceleration steps under centrifugal force,
and combinations thereof.
BACKGROUND
[0003] Current strategies in regenerative medicine aim towards
replacing tissue that undergoes an increased apoptosis rate. That
means that within the organ there is a net loss of functional cells
because more cells are dying than are being replaced. Therefore,
the transfer of regenerative cells, e.g., stem cells and progenitor
cells, from one location to the site of renewal is a therapeutic
approach to restore the organ back to an equilibrium. Research at
our laboratories has shown that stem cells and regenerative cells
are present in every organ, primarily located in the vascular and
perivascular space with the early progenitor cells in the vessel
wall attached to the lamina elastica interna. One population of
these cells is able to replace the stroma of an organ, the other
part of these cells is capable to differentiate into the respective
parenchym of the specific organ. Each organ can be compared to a
house, where the stroma made from fibroblasts and consisting of
extracellular matrix can be compared to walls of bricks and mortar
in a house, the piping in a house corresponds to blood vessels of
the organ and nerves represent the electrical wiring in the walls.
Inside these houses, in each organ one has a certain type of
inhabitants, such as liver cells, heart cells, bone cells,
cartilage or fat cells, also called the parenchym of an organ.
[0004] In order to restore function to a dysfunctional organ, it is
important to provide both the stroma, that means the housing that
form the walls of the organ and the inhabitants, which are the
specific parenchymal cells.
[0005] A simple means to recover the regenerative cells capable of
restoring organ function is to dissociate them from subcutaneous
fat tissue, because it is rich in blood vessels and the removable
adipose tissue is not essential for life. Most people are capable,
even happy, to donate several grams of those tissues.
[0006] Autologous grafting of tissue harvested by lipoaspiration is
a common procedure in cosmetic surgery for both small (e.g.,
nasolabial folds) and large (buttocks or breast) volume filling
applications. The primary benefits of this procedure termed
"autologous fat grafting" are lower cost versus synthetic fillers
and no immune rejection since the patient's own tissue is used.
Currently, multiple methods of lipoaspirate collection and
processing are employed to obtain tissue for grafting. Factors that
determine clinical outcomes following autologous fat grafting have
not been fully elucidated. However, it is widely recognized that
improving the persistence of the graft is an area of significant
need.
SUMMARY
[0007] Transferred fat tissue or adipose cells recovered from one
location in the body and transferred to another continue to have an
aerobic metabolism. Without adequate blood supply these cells
undergo necrosis, apoptosis, and autophagy within 24 to 48 hours
and die. The problem with traditional fat grafting, where fat is
taken out by liposuction from one location in the body and
re-injected at another location, is that a great fraction of cells
transferred do not survive and the dying cells can cause
considerable local inflammation. Persistence and regenerative
potential of the fat graft is not correlated with the content of
lipid-filled adipocytes in the graft, but instead with the content
of regenerative cells such as stem cells and with the administered
extracellular matrix. Post-grafting loss of mature adipocytes is
high due to the trauma of harvest and re-administration and to the
ischemic nature of the graft environment. In contrast, stem and
regenerative cells are more resistant to these factors and thus
contribute substantially to long term graft viability and
regenerative potential.
[0008] The present document is based on an improved method for
volume filling of subcutaneous tissue or other connective tissue
structures that need a volume build up. The method includes
transfer of a cell-enriched matrix that has a reduced lipid
content, but an increased concentration of regenerative cells. It
is known that connective tissue has a considerably long tolerance
to ischemia since it has a significantly lower metabolism compared
to adipocytes cells. Therefore it is the aim of the present
invention to provide method and apparatus for a long-term stable
volume filling with a cell-cell-enriched matrix. This results in
greater survival of the tissue when it is transplanted to a new
location. Accordingly as described herein, neovascularization from
the tissue resident stem cells provides a greater viability of the
transplanted graft. An additional improvement to this method is to
re-apply a mixture of the cell-enriched matrix together with
dissociated regenerative cells recovered by the method described
herein that includes enzymatic dissociation of lipoaspirate in a
heated centrifuge by acceleration and deceleration in an inverted
rotor.
[0009] Accordingly, the present document provides novel methods for
preparation and recovery of a cell-enriched matrix, improved
recovery of regenerative cells from their subcutaneous location by
means of the same apparatus as used for the recovery of the matrix,
and the combination of cell-enriched matrix and regenerative cells
for enhanced neovascularization and better survival of the
transplanted volume filling graft tissue.
[0010] The present document also provides provide cost effective
means for recovery of regenerative cells, which are defined as
early mesenchymal cells plus the whole range of progenitor cells,
from their location in subcutaneous adipose tissue. Pre-processing
and reducing the content of lipid-filled cells from the initial
lipoaspirate is an effective method to save costly enzymes such as
collagenase and neutral protease. The methods include a two-step
approach where the amount of lipid-filled cells in the lipoaspirate
is reduced and a cell-enriched matrix is recovered, and then
subjecting the cell-enriched matrix with reduced lipid content to
an enzymatic and mechanical process by using also increased
temperature from a heated centrifuge with a reconfigurable rotor
and repeated cycles of acceleration and deceleration to recover a
regenerative cellular preparation at optimized cost.
[0011] Currently known methods to process subcutaneous fat with the
aim to obtain a processed lipoaspirate apply just centrifugation.
As shown herein, processing by centrifugation alone can increase
the cellular yield of processed tissue. However, extruding the
adipose tissue before the centrifugation step significantly
increases the cellular content of the processed lipoaspirate
material, referred to as the cell-enriched matrix.
[0012] Processing of subcutaneous tissue can be performed as
described herein to yield a cell-enriched matrix, which primarily
consists of collagen, laminin, elastin and other proteoglycans of
the extracellular matrix and tissue resident cells, including stem
and progenitor cells, collectively "regenerative cells" or
"regenerative platform," still bound in the tissue. Typically, a
cell-enriched matrix contains 90% or more of the regenerative cells
bound in their tissue location.
[0013] In one embodiment, the present document provides methods and
an apparatus for preparing and recovering a cell-enriched
matrix.
[0014] In one embodiment, the present document provides methods and
an apparatus for preparing and recovering regenerative cells from
the cell-enriched matrix.
[0015] In one embodiment, cellular compositions are provided that
include regenerative cells isolated as described herein, or
cellular compositions containing both a cell-enriched matrix and
regenerative cells. The cell-enriched matrix prepared as described
herein has a reduced lipid content, but an increased concentration
of regenerative cells. It is known, that connective tissue has a
considerably long tolerance to ischemia since it has a
significantly lower metabolism compared to adipocytes cells.
Cellular compositions described herein can enhance
neovascularization of grafts and increase long term survival of the
graft. Cellular compositions containing a combination of the
cell-enriched matrix and regenerative cells are particularly useful
for enhancing neovascularization of grafts and increasing long term
survival of the graft.
[0016] In one embodiment, this document features a method for
recovering cell-enriched matrix from tissue. The method includes
extruding a tissue sample that contains a suspension of adipose
tissue pieces in an aqueous fluid through an ostium and
centrifuging the extruded tissue sample to isolate a cell-enriched
matrix. The ostium can be from 1 to 5 mm in diameter. The extruded
tissue sample is centrifuged for at least five minutes at a minimum
of 400.times.g, preferably at higher g force up to 1200.times.g
(e.g., 400.times.g to 1200.times.g). Cells can be recovered from
the cell-enriched matrix as described herein.
[0017] In another aspect, this document features a method for
recovering cells from tissue. The method includes providing an
extruded tissue sample housed in a container adapted for a
centrifuge, the tissue sample comprising a suspension of tissue
pieces in an aqueous fluid; subjecting the sample to at least one
acceleration and deceleration step using centrifugal force applied
through a rotating element, wherein the rotating element comprises
a shaft and one or more arms that extend from the shaft, wherein
(i) the one or more arms are supported from the shaft in such a
manner that when the shaft rotates, the one or more arms swing
upward and outward relative to the shaft or (ii) the one or more
arms are supported at a fixed angle, wherein the containers
attached to said arms are held in such a position that
gravitational force on material is opposite of applied centrifugal
force, wherein said applied centrifugal force ranges from about 50
g to about 4000 g. The temperature of the sample can be maintained
between 32.degree. and 42.degree. C. One or more enzymes (e.g.,
proteases such as collagenases or neutral proteases, or other
enzymes as described herein) also can be included.
[0018] In one embodiment, this document features a method for
recovering a regenerative platform from a tissue sample (e.g.,
lipoaspirate, adipose tissue, and combinations thereof). The method
includes providing a tissue sample housed in a first tissue
collection container adapted for an automated tissue processing
unit, wherein the automated tissue processing unit comprises a
removable rotating apparatus comprising at least two cavities,
wherein each cavity is configured for detachably inserting a tissue
collection container within the cavity wherein the tissue sample
comprises a suspension of tissue pieces in an aqueous fluid; and
subjecting the tissue sample to at least one round of
centrifugation of at least 400.times.g for at least about 5 minutes
using the automated tissue processing unit, thereby separating a
cell-enriched matrix from the tissue sample, wherein the
cell-enriched matrix comprises a regenerative platform. The method
further can include extruding the tissue sample through an orifice
prior to placing the tissue sample into the automated tissue
processing unit. The cell-enriched matrix can have a higher
concentration of the regenerative platform compared to an otherwise
corresponding method absent the extruding the tissue sample through
an orifice. The method further can include transferring the tissue
sample concentrate from the first tissue collection container into
a second collection container by a closed system method. In some
embodiments, at least one protease can be added to the second
collection container. The cell-enriched matrix can be subjected to
at least two rounds of acceleration, wherein each round of
acceleration is followed by a round of deceleration, thereby
disaggregating the cell-enriched matrix. In some embodiments, the
cell-enriched matrix can be filtered to obtain an injectable
regenerative platform.
[0019] In any of the methods described herein, the method further
can include administering at least a portion of the injectable
regenerative platform into a subject at an injection site, whereby
the injection alters an area at or near the injection site.
[0020] This document also features a method for disaggregating a
cell-enriched matrix having a regenerative platform therein,
wherein the method comprises providing a cell-enriched matrix
housed in a second tissue collection container adapted for an
automated tissue processing unit, wherein the tissue collection
container comprises at least one protease; and subjecting the
cell-enriched matrix to at least two rounds of acceleration,
wherein each round of acceleration is followed by a round of
deceleration, and wherein at least two rounds of acceleration and
deceleration are performed at a rate of at least 10.times.g thereby
disaggregating the cell-enriched matrix. The method further can
include filtering, or filtering and concentrating, the
disaggregated cell-enriched matrix to obtain an injectable
regenerative platform.
[0021] This document also features a removable rotating apparatus
comprising at least two cavities, wherein each cavity is configured
for detachably inserting a tissue collection container within the
cavity, wherein the removable rotating apparatus is configured to
rotate within an automated tissue processing unit for separating a
cell-enriched matrix from a tissue sample. The removable rotating
apparatus can include a radio-frequency identification (RFID) tag
attached thereto that allows the removable rotating apparatus to be
identified by the automated tissue processing unit. Alternatively,
the type of removable rotating apparatus may be identified based on
the amount of electrical current required to accelerate the
apparatus during the acceleration phase. The removable rotating
apparatus can include autoclavable materials.
[0022] In another aspect, this document features an automated
tissue processing unit for isolating a cell-enriched matrix from a
tissue sample. The automated tissue processing unit can include a
removable rotating apparatus comprising at least two cavities,
wherein each cavity is configured for detachably inserting a tissue
collection container within the cavity. The automated tissue
processing unit can include a temperature control device. The
automated tissue processing unit can be configured to have at least
two stop-start intervals of acceleration. The removable rotating
apparatus can have at least one pre-determined specification that
allows the automated tissue processing unit to identify the
removable rotating apparatus.
[0023] In another aspect, a modified centrifuge is provided that
can be used to perform at least two series of rapid acceleration
and deceleration steps under centrifugal force. Such steps can be
performed in a thermally regulated environment (e.g., 35-42.degree.
C.) in the presence of one or more enzymes (e.g., a collagenase and
a neutral protease) to enhance the degradation of the extracellular
matrix and release of cells. Centrifugation can be used to recover
cells released from the extracellular matrix. The methods and
apparatus described herein can be used to process any human or
animal tissue that contains blood vessels. The methods and
apparatus are particularly useful for recovering cells from adipose
tissue (e.g., subcutaneous or intra-abdominal adipose tissue),
which is rich in vascularization and easy to recover from a
subject.
[0024] This document also provides a method for recovering cells
from tissue. The method includes providing a tissue sample housed
in a container adapted for a centrifuge, the tissue sample
including a suspension of tissue pieces in an aqueous fluid; and
subjecting the tissue sample to a plurality of acceleration and
deceleration steps using centrifugal force. The tissue sample can
include human tissue or animal tissue, and can contain blood
vessels. The tissue sample can be adipose tissue such as
lipoaspirate. The method can include maintaining a temperature of
from 26.degree. C. to 42.degree. C. inside the container while
subjecting the tissue sample to the plurality of acceleration and
deceleration steps. The tissue sample can be subjected to the
plurality of acceleration and deceleration steps in the presence of
one or more enzymes (e.g., a collagenase, other protease, or a
mixture thereof).
[0025] In some embodiments, each of the acceleration steps can be
performed for 5 to 20 seconds and each of the deceleration steps
can be performed for 3 to 20 seconds. The tissue sample can be
subjected to the plurality of acceleration and deceleration steps
for 5 minutes to 180 minutes (e.g., 20 minutes to 60 minutes). In
one embodiment, the tissue sample is subjected to at least three
cycles of acceleration to 200.times.g and deceleration to 1.times.g
per minute for 30 minutes.
[0026] In another aspect, this document features a method for
recovering cells from tissue. The method includes providing a
tissue sample housed in a container adapted for a centrifuge, the
tissue sample including a suspension of tissue pieces in an aqueous
fluid; subjecting the sample to a plurality of acceleration and
deceleration steps using centrifugal force applied through a
rotating element, wherein the rotating element comprises a shaft
and one or more arms that extend from the shaft, wherein (i) the
one or more arms are supported from the shaft in such a manner that
when the shaft rotates, the one or more arms swing upward and
outward relative to the shaft or (ii) the one or more arms are
supported at a fixed angle, wherein the containers attached to the
arms are held in such a position that gravitational force on
material is opposite of applied centrifugal force, wherein the
applied centrifugal force ranges from about 50 g to about 4000 g.
Each of the acceleration steps can be performed for 5 to 20
seconds. Each of the deceleration steps can be performed for 3 to
20 seconds. The tissue sample can be subjected to the plurality of
acceleration and deceleration steps for 5 minutes to 180 minutes
(e.g., 20 minutes to 60 minutes). In one embodiment, the tissue
sample is subjected to at least three cycles of acceleration to
200.times.g and deceleration to 1.times.g per minute for 30
minutes.
[0027] This document also features a method for recovering
regenerative cells from tissue. The method includes providing a
tissue sample housed in a container adapted for a centrifuge, the
tissue sample comprising a suspension of tissue pieces in an
aqueous fluid; subjecting the sample to a plurality of acceleration
and deceleration steps using centrifugal force; and centrifuging
the sample at 400 to 4000.times.g to isolate cellular components.
The sample, when subjected to centrifugation at 400 to
4000.times.g, can be housed within a container that includes an
elongated cylindrical central portion; a first end portion
integrally formed with the central portion; and a second open end
portion integrally formed with the central portion, wherein the
first end portion narrows down to a narrow opening, and comprises a
collection portion protruding from the end portion at the narrow
opening, wherein the collection portion is capable of receiving and
storing a liquid and comprises a removable plug to seal the first
end portion from the collection portion.
[0028] In any of the methods described herein, the container can
include a porous insert, wherein the porous insert is composed of a
biocompatible material and having a pore size ranging from 0.5 mm
to 5 mm, wherein the porous insert enhances the dissociation of
cells from the extracellular matrix of the tissue sample when the
tissue sample is subjected to said plurality of acceleration and
deceleration steps. The porous insert can be substantially
cylindrical in shape, an inverted substantially conical shape, or
can bisect the container into upper and lower portions.
[0029] In any of the methods described herein, the container can
include a plurality of particles, wherein the particles are at
least 100 micrometer in diameter and composed of one or more
biocompatible materials, wherein the particles enhance the
dissociation of cells from the extracellular matrix of the tissue
sample when the tissue sample is subjected to the plurality of
acceleration and deceleration steps. The plurality of particles can
include particles of different specific gravities or shapes.
[0030] In any of the methods described herein, the container can
include a shaft disposed vertically in the internal lumen of the
container, the shaft further including a plurality of arms disposed
along a length of the shaft and extending substantially radially
from the shaft into the lumen of the container, wherein the arms
enhance the dissociation of cells from the extracellular matrix of
the tissue sample when the tissue sample is subjected to the
plurality of acceleration and deceleration steps. The arms can be
of different shapes or sizes. The container can include a removable
lid, wherein the shaft is affixed to the removable lid. The shaft
can be rotatably affixed to the container. The shaft can be
moveable within the lumen of the container.
[0031] In another aspect, this document features a container or
container assembly that includes an elongated cylindrical central
portion; a first end portion integrally formed with the central
portion; a second open end portion integrally formed with the
central portion; and a port extending radially outward from the
elongated cylindrical portion, wherein the first end portion
narrows down to a narrow opening, and includes a collection portion
protruding from the end portion at the narrow opening, wherein the
collection portion is capable of receiving and storing a fluid and
comprises a removable plug to seal the first end portion from the
collection portion. The removable plug can allow fluid to flow from
the end portion into the collection portion upon centrifugal force,
pressure, dissociation with an enzyme, or physical removal. The
collection portion can be detachable from the first end portion.
The collection portion can include an aqueous fluid. The second
open end includes a mating portion.
[0032] This document also features a container assembly that
includes a first container; a second container; and a coupling
device adapted to couple the first container to the second
container. The first container is described above. The second
container includes an elongated cylindrical central portion, a
closed end portion integrally formed with the central portion, and
an open end portion integrally formed with the central portion,
wherein the open end portion of the second container comprises a
mating portion; and the coupling device comprising a tubular
central portion with first and second open ends and a porous insert
extending horizontally across the coupling device, wherein each
open end of the coupling device comprises a mating portion, wherein
the porous insert has a pore size of 40 to 500 .mu.m. The coupling
device further includes a port extending radially outward from the
tubular central portion, wherein one mating portion of the coupling
device is attached to the mating portion of the first container and
the other mating portion of the coupling device is attached to the
mating portion of the second container. The port can include a
porous insert.
[0033] The first and second containers can be pre-assembled,
wherein an interior space defined by the first container and the
second container is at least partially under vacuum.
[0034] This document also features a container that includes an
elongated cylindrical central portion defining an internal lumen; a
first end portion integrally formed with the central portion; a
second open end portion integrally formed with the central portion;
a shaft disposed vertically in the internal lumen; and a plurality
of arms disposed along a length of the shaft and extending in a
substantially radial direction from the shaft into the internal
lumen. The plurality of arms can be of different shapes or sizes.
The container further can include a removable lid that attaches to
the second open end portion, wherein the shaft is affixed to the
removable lid. The shaft can be rotatably affixed to the container.
The shaft can be moveable within the lumen of the container.
[0035] In another aspect, this document features a kit that
includes any of the containers or container assemblies described
herein. The kit further one or more cell separation reagents.
[0036] In another aspect, this document features a method to
increase the cellular content in a processed lipoaspirate with the
aim to recover a cell enriched matrix for re-application to a
patient, the method includes extruding the lipoaspirate through an
orifice of a defined diameter in the range of 1-5 mm, and then
subjecting the extruded lipoaspirate to a continuous centrifugation
step of at least five minutes and a g-force of a minimum of
400.times.g. The centrifugation step can include centrifugal force
of up to 2000.times.g. The centrifugation step can include
centrifugal force of about 1200.times.g. The tissue sample can
include lipoaspirate, adipose tissue, and combinations thereof. The
centrifugation can be performed using a fixed angle, horizontal
rotor.
[0037] In another aspect, this document features a method of
facilitated recovery of regenerative cells from adipose tissue
comprising accelerating and decelerating the tissue in the presence
of proteolytic enzyme within a centrifuge, whereby the container
for the tissue is inverted. The interior of the centrifuge can be
temperature controlled. One or more cycles per minute of
acceleration and deceleration can be applied to the tissue. The
proteolytic enzyme can be a collagenase, a neutral protease or
both. A combination of collagenase and a neutral protease can be
used together with increased temperature and agitation by
centrifugal acceleration and deceleration in an inverted rotor for
facilitated recovery of regenerative cells from adipose tissue.
[0038] This document also features a method of cost effective
recovery of regenerative cells from adipose tissue comprising
providing a cell enriched matrix; accelerating and decelerating the
cell-enriched matrix in the presence of proteolytic enzyme within a
centrifuge, whereby the container for the tissue is inverted. The
interior of the centrifuge can be temperature controlled. One or
more cycles per minute of acceleration and deceleration can be
applied to the cell-enriched matrix. A combination of collagenase
and a neutral protease can be used together with increased
temperature and agitation by centrifugal acceleration and
deceleration in an inverted rotor for facilitated recovery of
regenerative cells from cell-enriched matrix.
[0039] This document also features a composition containing a
cell-enriched matrix prepared as described herein together with a
regenerative cell preparation prepared as described herein for
injection into a patient.
[0040] In another aspect, this document features a removable
rotating apparatus comprising at least two cavities, wherein each
cavity is configured for detachably inserting a tissue collection
container within the cavity, wherein the removable rotating
apparatus is configured to rotate within an automated tissue
processing unit for separating a cell enriched matrix from a tissue
sample. The removable rotating apparatus comprises a
radio-frequency identification (RFID) tag attached thereto that
allows the removable rotating apparatus to be identified by the
automated tissue processing unit. The removable rotating apparatus
can include autoclavable materials.
[0041] This document also features an automated tissue processing
unit for isolating a cell enriched matrix from a tissue sample,
wherein the automated tissue processing unit comprises a removable
rotating apparatus comprising at least two cavities, wherein each
cavity is configured for detachably inserting a tissue collection
container within the cavity. The automated tissue processing unit
comprises a temperature control device. The removable rotating
apparatus has at least one pre-determined specification that allows
the automated tissue processing unit to identify the removable
rotating apparatus.
[0042] In another aspect, this document features a method for
recovering cells from adipose tissue. The method including
extruding lipoaspirate through an ostium; centrifuging the extruded
lipoaspirate to produce a cell enriched matrix; and subjecting the
cell enriched matrix to a plurality of acceleration and
deceleration steps using centrifugal force to recover regenerative
cells from the cell enriched matrix. The method further can include
maintaining a temperature of from 26.degree. C. to 42.degree. C.
inside the container while subjecting the tissue sample to the
plurality of acceleration and deceleration steps. The tissue sample
can be subjected to the plurality of acceleration and deceleration
steps in the presence of one or more enzymes (e.g., a collagenase,
other protease, or a mixture thereof).
[0043] In another aspect, this document features a method for
recovering cells from tissue. The method includes providing a
tissue sample housed in a container adapted for a centrifuge, the
tissue sample comprising a suspension of tissue pieces in an
aqueous fluid; subjecting the sample to at least one acceleration
and deceleration step using centrifugal force applied through a
rotating element, wherein the rotating element comprises a shaft
and one or more arms that extend from the shaft, wherein (i) the
one or more arms are supported from the shaft in such a manner that
when the shaft rotates, the one or more arms swing upward and
outward relative to the shaft or (ii) the one or more arms are
supported at a fixed angle, wherein the containers attached to said
arms are held in such a position that gravitational force on
material is opposite of applied centrifugal force, wherein said
applied centrifugal force ranges from about 50 g to about 4000 g.
The temperature of the sample can be maintained between 32.degree.
and 42.degree. C. The tissue sample further can include one or more
proteases.
[0044] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the exemplary methods and materials are described below.
All publications, patent applications, patents, Genbank.RTM.
Accession Nos, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present application, including definitions, will control. The
materials, methods, and examples are illustrative only and not
intended to be limiting.
[0045] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF DRAWINGS
[0046] FIG. 1 is a perspective view of an apparatus for
dissociation, separation and recovery of cells from a tissue sample
according to an embodiment described herein.
[0047] FIG. 2 is a graph of different time and energy cycles during
the dissociation phase and combinations thereof.
[0048] FIGS. 3A-3E are perspective views of different containers
adapted for a centrifuge.
[0049] FIG. 4 is a perspective view of a container assembly
according to one embodiment described herein.
[0050] FIG. 5 is a side cross-sectional view of one embodiment of
the apparatus of FIG. 1.
[0051] FIG. 6 is a bar graph of the number of adherent cells
obtained after processing using an embodiment of the apparatus of
FIG. 1 or after processing with a shaking incubator.
[0052] FIG. 7 is a bar graph of the number of adherent cells
obtained after processing using an embodiment of the apparatus of
FIG. 1 and containers of FIG. 3A, 3D, or 3E, or after processing
using a shaking incubator.
[0053] FIG. 8 is a top view of a removable rotating apparatus that
may be inserted into an automated tissue processing unit.
[0054] FIG. 9 is a side view of a removable rotating apparatus that
may be inserted into an automated tissue processing unit.
[0055] FIG. 10 is a graph of the number of adherent cells/g tissue
obtained from adipose tissue that was treated as follows: not
centrifuged, centrifuged, or extruded then centrifuged.
[0056] FIG. 11 is a graph of the number of cells/g tissue obtained
from adipose tissue that was not extruded, extruded through an
emulsion needle (SS extruded), or Luer extruded, and then either
not centrifuged or centrifuged at 1200.times.g. For extrusion the
tissue was passed 5.times. across the extrusion device using
syringes
[0057] FIG. 12 is a graph of the number of adherent cells/g tissue
obtained from adipose tissue after 5, 10, or 20 minutes of
centrifugation.
[0058] FIG. 13 is a graph of the number of adherent cells/g tissue
obtained from adipose tissue after no centrifugation,
centrifugation at 400.times.g for 30 minutes, or 1200.times.g for
30 minutes.
[0059] FIG. 14 is a schematic of a method to increase the
concentration of adipose derived regenerative cells (ADRC) in a
tissue preparation for grafting. Tissue is first processed to
concentrate ADRC in the cell-enriched matrix (CEM). One-half of the
CEM is processed to yield isolated ADRC, which are then combined
with the remaining CEM to further increase the concentration of
ADRC in the graft.
[0060] FIG. 15 is a schematic of a method to increase the
efficiency of enzyme and disposable utilization in processing of
adipose tissue or lipoaspirate to obtain ADRC. Tissue is first
processed to concentrate ADRC in CEM, which is then combined and
processed to yield ADRC.
[0061] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0062] In general, this document is based on methods and apparatus
for recovery of cells from tissues, including human and animal
tissues such as canine, feline, equine, bovine, ovine, or porcine
tissues. The methods and apparatus described herein are
particularly useful for recovery of cells from adipose tissue
obtained from, for example, liposuction (i.e., lipoaspirate),
including suction assisted, vapor assisted, or ultrasound assisted
liposuction, and combinations thereof. For instance, the methods
and apparatus described herein can be used to isolate stem cells,
progenitor cells, hematopoietic cells, or fully differentiated
cells from adipose tissue.
[0063] The methods and apparatus described herein can be used on
site to prepare cellular compositions for administration to a
patient (e.g., autologous administration). For example, the methods
and apparatus described herein can be used to recover regenerative
cells from a patient, referred to herein as a regenerative
platform, that can be prepared for administration and then
administered (e.g., injected or surgically implanted) back to the
patient from which the cells were recovered. In some embodiments,
the cells can be loaded into a delivery device such as a syringe,
for injection into the recipient by, for example, subcutaneous,
intravenous, intramuscular, or intraperitoneal techniques. For
example, the regenerative cells can be injected into blood vessels
for systemic or local delivery, into tissue (e.g., cardiac muscle
or skeletal muscle), into the dermis (subcutaneous), into tissue
space (e.g., pericardium or peritoneum), or other location.
Injection of the regenerative platform can result in an area near
the injection site being augmented, repaired, having reduced
inflammation, reduced pain, and combinations thereof. In some
embodiments, one or more additives are added to the cells before
administration. For example, the cells can be mixed with other
cells, biologically active compounds, biologically inert compounds,
demineralized bone, a matrix or other resorbable scaffold, one or
more growth factors, or other additive that can enhance the
delivery, efficacy, tolerability, or function of the cell
population.
[0064] The methods and apparatus also can be used to prepare
cellular compositions for growth studies, gene expression studies,
differentiation studies, or other research purposes. In addition,
the methods and apparatus described herein can be used to recover
regenerative cell populations (e.g., stem cells) such that the
cells can be banked, for example, by cryopreserving the cells with
an appropriate medium. For further reference, see U.S. Patent
Application Publication No. 20100285588-A1.
[0065] In one embodiment, the methods described herein use a
plurality of, i.e., two or more, acceleration and deceleration
steps under centrifugal force to enhance the dissociation of the
cells (e.g., regenerative cells such as stem or progenitor cells)
from the extracellular matrix of the tissue. One acceleration and
deceleration step under centrifugal force can be referred to as one
round of centrifugation. In some embodiments, one or more of the
following also can be used to enhance recovery of the cells:
mechanical disruption, mechanical agitation, maintaining the
temperature above room temperature (>26.degree. C.) and at or
below 42.degree. C. (e.g., about 37.degree. C. to 40.degree. C.),
using enzymes to degrade the tissue, and separating different
components of the tissue based on physical characteristics such as
density, specific weight, and solubility. In some embodiments, the
tissue is mechanically disrupted (e.g., by extrusion) before
subjecting the tissue to two acceleration and deceleration steps
under centrifugal force. In some embodiments, after mechanical
disruption of the tissue, a first acceleration and deceleration
step is performed under centrifugal force to separate the tissue
into three general layers (i.e., an aqueous layer, a cell-enriched
matrix containing the regenerative platform, and a lipid layer).
The cell-enriched matrix can be removed, and subjected to a second
acceleration and deceleration step under centrifugal force. In some
embodiments, one or more enzymes (e.g., proteases) are added to the
cell-enriched matrix before subjecting the cell-enriched matrix to
the second acceleration and deceleration step. Such a process
reduces the sample volume such that a smaller amount of enzymes is
required to process the tissue. In some embodiments, a portion of
the cell-enriched matrix is subjected to a second acceleration and
deceleration step under centrifugal force, and the regenerative
cells isolated from the cell pellet. The cell isolated from the
portion of the cell-enriched matrix then can be combined with the
cell-enriched matrix that has not been subjected to further
centrifugation.
[0066] Using a reconfigurable centrifuge rotor that can be
configured in an inverted position or in a swinging bucket
configuration as described below is particularly useful in the
methods described herein. When in the inverted configuration,
centrifugal forces drive contents toward the outer, higher portion
of a sample container, and at rest these contents return to the
inner, lower portion of the sample container. Thus, by repeated
cycles of acceleration and deceleration, an inverted rotor provides
a means for agitation of a sample in a way, that at rest with
gravitation and no acceleration forces, the contents of a container
reside and move towards the center and axle of a centrifuge while
with acceleration, the contents follow the centrifugal forces and
go outward. When tissue is combined with a proteolytic enzyme
solution as described herein, and placed in a container in the
reconfigurable rotor in inverted position, repeated cycles of
acceleration and deceleration facilitate the enzymatic dissociation
of the tissue. When the reconfigurable rotor is configured in a
typical swinging bucket rotor configuration, however, the contents
of the sample container concentrate at the bottom and agitation
therefore is reduced.
[0067] In order to significantly improve the recovery of
regenerative cells and shorten the onsite application of the
methods described herein in the operating theater, increasing the
ambient temperature inside the centrifuge chamber during repeated
cycles of acceleration and deceleration to 35 to 42.degree. C.
(e.g., 40.degree. C.) increases the speed of enzymatic dissociation
by several fold, compared to room temperature
[0068] The methods and apparatus described herein increase the
yield of cells recovered from the tissue sample relative to other
methods in which a plurality of acceleration and deceleration steps
under centrifugal force are not utilized. The increased yield of
cells recovered using the methods and apparatus described herein
are surprising in view of the findings by Kurita et al., Plast.
Reconst. Surg., 121:1033-1041 (2008) in which centrifugation alone
at constant speed is not sufficient to release increased numbers of
viable stem cells from the extracellular matrix.
[0069] FIG. 1 is a perspective view of one embodiment of a
centrifuge 1 having an inner chamber separated by a wall 2 (e.g.,
metallic wall) thereby creating an inner container 4. Typically,
the diameter of the inner container 4 is 20-40 cm, depending on the
size of the containers used for the cellular preparation. Within
inner container 4 is an axle 6 driven by a motor 8 to rotate axle
6. The motor 8 typically is located below inner container 4. A
controller 10 can turn the motor 8 on or off, and also can serve to
regulate temperature inside inner container 4 as described below.
Motor 8 through axle 6 turns rotor 12, which has two (as depicted)
or more arms, each of which is capable of receiving and gripping
container 16 in a firm link to the rotor arm 14. In one embodiment,
rotor arm 14 can swing up and down based on the centrifugal forces
exerted through motor 8 via axle 6, and depending on their speed,
change their position from a vertical position to a fully 90 degree
position when a certain g force (e.g., 20-30 g) is exceeded by the
rotations per minute of the axle 6. In another embodiment, such as
that depicted in FIG. 5, rotor arm 14 is set at a fixed angle
.theta. (e.g., an angle ranging from 1 degree to less than 90
degrees). For example, the angle .theta. can be fixed at 12
degrees. In such an embodiment, container 16 can be attached to
rotor arm 14 in an inverted orientation in which the top of
container 16 containing a removable lid is oriented near the center
by axle 6.
[0070] In some embodiments, the temperature of inner container 4
can be regulated such that the temperature of the inner container
ranges, for example, from 26 to 42.degree. C., 30 to 42.degree. C.,
35 to 42.degree. C., 35 to 40.degree. C., 37 to 40.degree. C. or
about 37.degree. C. The temperature can be regulated by any known
method, e.g., closed loop thermal feedback regulation. In one
embodiment, electrical resistance wires (not shown in FIG. 1) can
be wrapped around or embedded in the inner container 4 in order to
warm up the inner container 4 through a connection to electricity,
for example, via a cable. Such wires can be part of a heat pad or
embedded in a flexible polymer. In order to keep the temperature
constant, a temperature probe 18, which is operably linked to
controller 10, can be used to sense the temperature in the inner
container 4 and via controller 10, regulate the temperature within
the inner container 4. Maintaining the temperature at 35 to
42.degree. C. is particularly useful when one or more enzymes are
used, as temperatures below this range can slow the dissociation
process and temperatures above 42.degree. C. can damage cells.
[0071] Controller 10 can be programmed to, for example, control the
acceleration and deceleration steps, start and stop the motor 8,
and regulate temperature. Controller 10 connects to a power source
(e.g., through a plug or cable).
[0072] Controller 10 can be programmed to accelerate container 16
to achieve a g force of between 50.times.g and 4,000.times.g
inclusive, maintain that g force for a short period of time, and
decelerate the container 16 to 1.times.g within a short period of
time. Repetitive cycles of the acceleration/deceleration steps can
be applied over a time frame from 5 to 180 minutes (e.g., 5 to 120
minutes, 10 to 100 minutes, 20 to 60 minutes, 25 to 50 minutes, 30
to 45 minutes, about 30 minutes, or about 45 minutes). For example,
each acceleration step can be performed for 5 to 20 seconds and
each deceleration step can be performed for 3 to 20 seconds. In one
embodiment, at least three cycles of acceleration to 200.times.g
and deceleration to 1.times.g per minute can be performed for 30
minutes.
[0073] FIG. 2 depicts examples of various time cycles that can be
used to enhance the dissociation of cells from the extracellular
matrix in the tissue sample contained in container 8 as shown in
FIG. 1. Different patterns as depicted in FIG. 2 can be combined
such as intermittent on and off and certain accelerations in which
a certain g force is maintained over a longer period of time.
[0074] FIGS. 3A-3E depict containers, each of which is an exemplary
embodiment of the container 16 shown in FIGS. 1 and 6. The
containers in FIGS. 3A-3E are adapted for use in a centrifuge
(e.g., a centrifuge depicted in FIG. 1 or FIG. 6). The containers
have an insert that can aid in the dissociation of the cells from
the extracellular matrix of the tissue when the containers are
subjected to a plurality of acceleration and deceleration steps
under centrifugal force, such as the centrifugal forces imparted by
centrifuge 1 of FIG. 1. In each of FIGS. 3A-3E, container 300
includes an elongated cylindrical central portion 302, a closed end
portion 304 integrally formed with the central portion, and an open
end portion 306 integrally formed with the central portion defining
an interior lumen 308. Closed end portion 304 can be substantially
flat, rounded, hemispherical, conical, or any other appropriate
shape. In some embodiments, the container 300 can have a length of
approximately 10-12 cm. In some embodiments, the container 300 can
have a volume of approximately 50-60 ml.
[0075] The open end portion 306 includes a mating portion 310
(e.g., a threaded portion) that is formed to accept a removable cap
312. The removable cap 312, when attached onto the mating portion
310, substantially encloses and seals the interior lumen 308 of the
container 300. In some implementations, the cap 312 may be attached
to the open end by threads, friction (e.g., a snap-on cap), by
clamping, by magnetic attraction, by a vacuum seal, or by any other
appropriate mechanism by which a vessel can be reversibly
sealed.
[0076] In FIG. 3A, container 300 includes an inverted substantially
conical insert 320. The conical insert 320 is formed as an inverted
hollow cone within the interior lumen 308. The inverted
substantially conical insert 320 is substantially coaxial with the
elongated cylindrical central portion 302, with a conical sidewall
322 that extends from a vertex 324 proximal to the enclosed end
portion 304 to a base 326 proximal the open end portion 306.
[0077] The inverted substantially conical insert 320 is composed of
a biocompatible material and is porous. Non-limiting examples of
biocompatible materials include polyamides (e.g., Nylon);
polyesters such as polycaprolactone; polystyrene; polypropylene;
polyacrylates; polyvinyl compounds; polycarbonate; polyketones such
as polyetheretherketone (PEEK); polytetrafluoroethylene (PTFE,
Teflon); thermanox; nitrocellulose; poly(ortho esters);
polyurethane; stainless steel; titanium; or titania (titanium
dioxide). The pore size of the insert can range from 0.5 mm to 5 mm
(e.g., 0.7 to 1.5 mm, 0.7 to 1.2 mm, 0.9 to 1.1 mm, 0.9 to 1.5 mm,
0.9 mm to 2.0 mm, 1 to 3 mm, 2 to 4 mm, 3 to 5 mm). In some
embodiments, the inverted substantially conical sidewall 322 can be
a screen, lattice, mesh, net, perforated sheet, or other suitable
biocompatible porous substrate. In some embodiments, the inverted
substantially conical insert can be a mesh with 1 mm pores.
[0078] The base 326 of the conical insert 320 has a diameter that
is substantially the same as the diameter of the elongated
cylindrical portion 302 at the open end 306. As such, the conical
insert 320 is inserted into the interior volume 308 through the
open end 306 until the base contacts the rim of the open end 306.
The cap 312 is then removably affixed onto the open end 306,
thereby substantially centering and affixing the conical insert 320
within the interior lumen 308. In some embodiments, the base 326
contains a flange that can be used to attach to the open end 308.
Base 326 also can be secured directly to the bottom surface of the
cap 312. Base 326 also can be secured directly to end 304.
[0079] In use, the inverted substantially conical insert 320 can be
inserted into the container 300. The inverted substantially conical
insert 320 can be filled with a tissue sample that includes a
suspension of tissue pieces in an aqueous fluid, such that fluids
and components of the tissue smaller than the pores are able to
pass through the conical insert 320 to be captured by the
cylindrical sidewall 304 and closed end 306.
[0080] In some embodiments, one or more proteases (e.g., one or
more collagenases such as type I and/or type II collagenases, a
neutral protease such as thermolysin, trypsin, or mixtures thereof)
can be added to a container 300 to enhance the dissociation of the
cells from the extracellular matrix of the tissue sample. For
example, a type I collagenase, a type II collagenase, and a dispase
can be used to enhance the dissociation of the cells from the
extracellular matrix.
[0081] The cap 312, once applied, seals the interior lumen 308 and
substantially affixes the conical insert 320 in position. The
fluids and tissues are urged through the pores of the substantially
conical insert 320 by the plurality of acceleration and
deceleration steps. For example, the container 300, with a tissue
sample loaded within the substantially conical insert 320, can be
attached onto the rotor arm 14 of the centrifuge 1 of FIG. 1. The
container 300 can then be accelerated and decelerated as discussed
in the description of FIGS. 1 and 2. Under the repeated cycles of
acceleration and deceleration, the tissue sample is urged in
various directions through the pores of the insert. This process
mechanically disrupts the tissue to enhance the release of the
cells from the extracellular matrix.
[0082] In FIG. 3B, container 300 include a substantially
cylindrical insert 340. The substantially cylindrical insert 340 is
formed as a cylinder within the interior lumen 308. The cylindrical
insert 340 is substantially coaxial with the elongated cylindrical
central portion 302, with a sidewall 344 that extends from closed
end portion 304 to open end portion 306. The substantially
cylindrical insert 340 is composed of a biocompatible material and
is porous. Examples of suitable biocompatible materials and pore
sizes are discussed above.
[0083] In use, the substantially cylindrical insert 340 can be
inserted into the container 300. The substantially cylindrical
insert 340 can be filled with a tissue sample containing a
suspension of tissue pieces in an aqueous fluid such that fluids
and components of the tissue smaller than the pores are able to
pass through the cylindrical insert 340 to be captured by the
elongated cylindrical central portion 302 and closed end 304. One
or more enzymes also can be added to the container as discussed
above. The cap 312, once applied, seals the interior volume 308 and
substantially affixes the cylindrical insert 340 in position. The
container 300 can then be accelerated and decelerated as discussed
in the description of FIGS. 1 and 2. Under the repeated cycles of
acceleration and deceleration, the tissue sample is urged in
various directions through the pores of the insert. This process
mechanically disrupts the tissue to enhance the release of the
cells from the extracellular matrix.
[0084] In FIG. 3E, the container 300 contains an insert 345 that
bisects the container into upper and lower portions. The insert is
composed of a biocompatible material and is porous. Examples of
suitable biocompatible materials and pore sizes are discussed
above. The insert can be held in place using, for example, a ring
made out of rubber. In use, the tissue sample containing the
suspension of tissue pieces in an aqueous fluid is loaded into the
upper or lower portion of the container and subjected to the
plurality of acceleration and deceleration steps to enhance the
dissociation of the cells from the extracellular matrix. One or
more enzymes also can be added with the tissue sample.
[0085] Referring now to FIG. 3C, a container 300 includes a
plurality of particles 350 within the interior lumen 308. The
pellets are at least 100 micrometers in diameter and are composed
of one or more biocompatible materials or coated with one or more
biocompatible materials. Non-limiting examples of biocompatible
materials include polyamides (e.g., Nylon); polyesters such as
polycaprolactone; polystyrene; polypropylene; polyacrylates;
polyvinyl compounds; polycarbonate; polyketones such as PEEK; PTFE;
thermanox; nitrocellulose; poly(ortho esters); polyurethane;
stainless steel; titanium; titania (titanium dioxide); and glass.
In some embodiments, the plurality of particles 350 can include
particles of different specific gravities, shapes, or surface
characteristics. In one embodiment, the plurality of particles 350
can include smooth polystyrene beads and particles with an iron
coating.
[0086] In use, a tissue sample containing a suspension of tissue
pieces in an aqueous fluid and the particles 350 are loaded into
the container 300 and sealed with the cap 312. In some embodiments,
one or more enzymes also are added to the container before sealing
with the cap. The container is then loaded into the centrifuge 1 of
FIG. 1 and is subjected to the plurality of acceleration and
deceleration steps, which cause the particles 350 to be agitated
and enhance the release of the cells from the extracellular
matrix.
[0087] FIG. 3D shows another example of a container 300 containing
an insert in which a shaft 370 is disposed vertically in the
internal lumen 308 of the container. The shaft 370 includes a
plurality of arms 372 disposed along a length of the shaft 370 and
extending substantially radially from shaft 370 into the lumen 308.
The arms 372 can be of different shapes and/or sizes as depicted in
FIG. 3D.
[0088] In some embodiments, shaft 370 can be affixed to removable
lid 312. Shaft 370 can be rotatably affixed to the container or
removable container lid 312. For example, the shaft 370 can be
rotatably affixed to, and extend through, the cap 312, such that
the shaft 370 can be gripped and rotated from outside the container
to agitate the tissue sample. In some embodiments, the shaft 370
can be rotatably affixed to the cap 312, and can be eccentrically
weighted such that the shaft 370 can rotate under the force of
gravity or centrifugation. In some embodiments, the shaft 370 can
be rotatably affixed to the cap 312, and one or more of the arms
372 can include a magnet such that the shaft 370 can be
magnetically coupled to a magnetic field external to the container.
By rotating the magnetic field relative to the container, the shaft
370 can be urged to rotate within the interior lumen 308 to agitate
the tissue sample. In some embodiments, shaft 370 can be moveable
vertically within lumen 308 such that the arms 372 can pass up and
down through the tissue sample. In some embodiments, the arms are
sharpened blades.
[0089] In one embodiment, a tissue sample containing a suspension
of tissue pieces in an aqueous fluid can be loaded into the
container 300 and the cap 312 with the shaft 370 attached is
affixed to seal the open end such that the arms 372 disposed along
a length of shaft 370 are inserted into the interior lumen 308 and
the tissue sample. One or more enzymes also can be added with the
tissue sample. The container 300 can then be subjected to a
plurality of acceleration and deceleration steps as discussed
herein. The shaft 370 and associated arms 372 can enhance the
dissociation of cells from the extracellular matrix of the tissue
sample when subjected to the plurality of acceleration and
deceleration steps.
[0090] FIG. 4 shows an example of a container assembly 400 of a
first container 300, second container 404, and a coupling device
406. In the embodiment depicted in FIG. 4, the assembly 400
includes the container 300 of FIG. 3A and a container 404. In some
embodiments, the container 300 can be any of the containers
depicted in FIGS. 3B-3E. The container 404 includes an elongated
cylindrical central portion 408; a first end portion 410 integrally
formed with central portion 408; and a second open end portion 412
integrally formed with central portion 408, defining an internal
lumen 414.
[0091] In some embodiments, a port 432 can extend radially outward
from the elongated cylindrical portion 408 and provide a fluidic
passage that extends from the interior lumen 414 to the outside.
Such a port also can include a porous insert 434. In some
embodiments, the porous insert can have pores of approximately 0.2
microns, which can allow air to pass but prevent contaminants from
entering the interior lumen 414.
[0092] The first end portion 410 narrows down to a narrow opening
416, and contains a collection portion 418 protruding from end
portion 410 at the narrow opening 416. The collection portion 418
is capable of receiving and storing a fluid and includes a
removable plug 420 to seal the first end portion 410 from the
collection portion 418. The removable plug 420 allows fluid to flow
from the end portion 410 into the collection portion 418 upon
centrifugal force, pressure, dissociation with an enzyme, or
physical removal. In some embodiments, the removable plug is a
valve that can be activated to provide access to the collection
portion. In some embodiments, the collection portion is detachable
from the first end portion.
[0093] In some embodiments, the collection portion 418 comprises an
aqueous fluid that is separated from the interior lumen 414 via
removable plug 420. For example, collection portion 418 can include
sterile saline, buffer, cell culture medium, one or more
biologically active compounds, one or more biologically inert
compounds, demineralized bone, a matrix or other resorbable
scaffold, one or more growth factors, or other additive that can
enhance the delivery, efficacy, tolerability, or function of the
cell population.
[0094] The second end portion can contain a mating portion (e.g., a
threaded portion) such that a cap can be attached to substantially
seal the container.
[0095] Container 404 can be removably connected to container 300
using coupling device 406, which includes a tubular central portion
422 with first and second open ends 424 and a porous insert 426
extending horizontally across coupling device 406. Each open end
424 includes a mating portion (e.g., a threaded portion). The
porous insert has a pore size of 40 to 500 micrometers and extends
horizontally across coupling device 406 such that porous insert 426
substantially separates the interior volume 308 from the interior
volume 414. In some embodiments, two or more porous inserts can be
disposed on top of one another. For example, a porous insert with
relatively larger pore sizes can be disposed more closely to the
container 300, while the porous inserts with relatively smaller
pore sizes can be disposed more closely to the container 404. As
such, fluids and particles flowing from the container 300 to the
container 404 can pass through progressively smaller pores as they
pass through the porous insert 426.
[0096] Coupling device 406 further can include a port 428 extending
radially outward from the tubular central portion 422 to provide a
fluidic passage from the interior of the coupling device. The port
can include a porous insert 430 with a pore size of 0.2 to 500
micrometers. In some embodiments, porous insert 430 can have pores
of approximately 0.2 microns, which can allow air to enter into the
interior of the coupling device 406, but filter out bacteria and
particulate matter than could contaminate the containers 300, 404
or the tissue sample.
[0097] One mating portion of the coupling device can be attached to
the mating portion of container 300 and the other mating portion of
the coupling device can be attached to the mating portion of
container 404. In embodiments in which the open end portions of
containers 300 and 404 are threaded, coupling device 406 is
threaded on each end to allow the containers 300 and 404 to be
threaded into coupling device 406 by their threaded portions. In
some embodiments, containers 300 and 404 are pre-assembled such
that an interior space defined by the first container and the
second container is at least partially under vacuum.
[0098] In use, the container 300 can be uncoupled from coupling
device 406 and loaded with a tissue sample, buffer (e.g., lactated
Ringer's), and optional enzyme. A cap (e.g., the cap 312) is
applied to removably seal the container 300. The container 300 and
the tissue sample within can be processed in the centrifuge 1 as
discussed above. After subjecting the sample contained within
container 300 to the plurality of acceleration and deceleration
steps as discussed above, the cap 312 can be removed and coupling
device 406 can be attached to container 300 and container 404. The
liquid components within container 300 can be forced into container
404 by inverting the container assembly and applying negative
pressure to port 432. For example, a small piece of tubing can be
attached to port 432 and suction applied using a syringe (e.g.,
with a Luer connection) to create a negative pressure in container
404 such that fluid and cells within the fluid in container 300 are
forced through the porous insert 426 and into the interior lumen
414 of container 404.
[0099] After transfer to container 404, container 300 can be
detached from the coupling device and the coupling device can be
detached from container 404. A cap can be removably attached to the
mating portion 424 to substantially seal the container 404. Cells
dissociated from the extracellular matrix then can be recovered
from other cellular components by centrifuging the container at 400
to 4000.times.g. In some embodiments, the cells are collected in
the collection portion 418 of container 404.
[0100] In some embodiments, the centrifugation at 400 to
4,000.times.g can be performed as a second program carried out
using centrifuge 1. For example, the second program can be
programmed into controller 10. The container 404 can be inserted
into the rotor arm 14 in a fixed angle embodiment in which the cap
is oriented toward the center of the rotor and the collection
portion 418 is oriented away. Container 404 can be centrifuged for
about 5 to 10 minutes with a g force of about 400.times.g to
4,000.times.g (e.g., 400 to 1,000.times.g). Centrifugation at such
g forces allows for separation and collection of cells at the
collection portion 418.
[0101] In some embodiments, one or more cell separation reagents,
including magnetic beads or antibodies or antigen binding fragments
thereof can be used in conjunction with the methods and apparatus
described herein. For example, antibodies having binding affinity
for a particular cell type can be used to recover cells of that
type from cells collected within the collection portion. In some
embodiments, such cell separation reagents are included within
container 300. In some embodiments, such cell separation reagents
are included within container 404.
[0102] In some embodiments, a tissue sample is subjected to one
acceleration and deceleration step under centrifugal force to
prepare a cell-enriched matrix, which is then subjected to one or
more acceleration and deceleration steps. For example, a tissue
sample housed in a tissue collection container can be centrifuged
to produce a cell-enriched matrix that includes a regenerative
platform therein. Regenerative platform refers to regenerative
cells such as stem cells, progenitor cells, and/or hematopoietic
cells within the concentrate. An example of such a cell preparation
is given in US 2010/0124563 A1. The tissue sample may have a
regenerative platform throughout the tissue sample; however,
centrifugation causes the tissue sample to form a cell-enriched
matrix having a concentrated amount of the regenerative platform
therein. For example, upon centrifugation of a tissue sample within
a tissue collection container, three general layers form (e.g., an
aqueous layer, a cell-enriched matrix having the regenerative
platform, and a lipid layer). The cell-enriched matrix is generally
located between the lipid layer and the aqueous layer. After the
extraction of the aqueous layer, the cell-enriched matrix can be
easily extracted from the tissue collection container (e.g., using
a closed system) for further use of the cell-enriched matrix.
[0103] An automated tissue processing unit can be used to
centrifuge the tissue sample. For example, an automated tissue
processing unit having a removable rotating apparatus therein,
where the removable rotating apparatus is configured to rotate
within the automated tissue processing unit, can be used to
generate the centrifugal force. The automated tissue processing
unit may include a temperature control device for controlling the
temperature within the unit.
[0104] FIG. 8 is a top view of a removable rotating apparatus 500
that may be inserted into an automated tissue processing unit (not
shown). A tissue collection container 502 may be detachably
inserted into the removable rotating apparatus 500 and held in
place by a locking mechanism 504 (e.g., snappable locking
mechanism). Here, the tissue collection container 502 snaps into
the cavity 506. In one non-limiting embodiment, the tissue
collection container 502 is customizable to snappably fit within
the cavity 506 and held in place by the snappable locking mechanism
504. The tissue collection container 502 is oriented so that the
opening 512 of the tissue collection container 502 is farthest from
the center. The formation of a cell-enriched matrix may form near
the opening 512, and the cell-enriched matrix may be easily
extracted from the tissue collection container 502 through the
opening without additional contamination to the cell-enriched
matrix.
[0105] The removable rotating apparatus 500 may have at least two
cavities (shown here as 506, 508) or may have up to about eight
cavities in another non-limiting embodiment. Each cavity 506, 508
may be in a horizontal orientation and may have a detachable
mechanism for inserting a tissue collection container 502 within
the cavity 506, 508. The detachable mechanism may be, but is not
limited to a snapping mechanism, Velcro, and the like, and
combinations thereof. In another non-limiting embodiment, the
removable rotating apparatus 500 may have or include autoclavable
materials, such that the removable rotating apparatus 500 is
configured to be autoclavable.
[0106] FIG. 9 is a side view of a removable rotating apparatus 500
that may be inserted into an automated tissue processing unit (not
shown). The tissue collection container 502 is shown within the
cavity 506 and held in place by the locking mechanism 504. It will
be appreciated that the removable rotating apparatus illustrated in
FIGS. 8-9 is not to scale or proportion and that certain features
of it may be exaggerated or distorted for illustrative
purposes.
[0107] The automated tissue processing unit may also have a
mechanism for identifying a particular removable rotating apparatus
by at least one pre-determined specification of the removable
rotating apparatus. In one embodiment, the removable rotating
apparatus may have an attached RFID tag. The RFID tag may be
scanned upon placement of the removable rotating apparatus into the
automated tissue processing unit for identification by the
automated tissue processing unit. In another embodiment, a
specification of the removable rotating apparatus within the
automated tissue processing unit may be measured and recorded, such
as, but not limited to, the power demand associated with
acceleration, weight, wind resistance, and combinations thereof.
The measured specification may be stored as part of a software
program and/or software package of the automated tissue processing
unit that enables the automated tissue processing unit to identify
the removable rotating apparatus by such specification data.
[0108] The tissue sample may be housed in a first tissue collection
container adapted for the automated tissue processing unit. The
tissue sample may have or include a suspension of tissue pieces in
an aqueous fluid. In one non-limiting embodiment, the tissue sample
may be extruded prior to placement of the tissue sample into the
first tissue collection container. The tissue sample may be
extruded between one and twenty times, or alternatively from about
two times to about ten times through an orifice ranging in diameter
from about 1 mm independently to about 4 mm, or alternatively from
about 1.5 mm independently to about 3 mm. Extruding the tissue
sample before subjecting it to a round of acceleration produces a
cell-enriched matrix that has a higher concentration of the
regenerative platform compared to an otherwise identical method
absent the extrusion of the tissue sample. As used herein with
respect to a range, "independently" means that any lower threshold
may be used together with any upper threshold to give a suitable
alternative range.
[0109] The tissue sample may be subjected to centrifugation to
achieve a g force ranging from about 200.times.g independently to
about 2000.times.g, or alternatively at least 400.times.g using the
automated tissue processing unit. The centrifugation may occur for
a time period ranging from about 3 minutes independently to about
60 minutes, or at least about 5 minutes. After the centrifugation,
a cell-enriched matrix may form.
[0110] The cell-enriched matrix can be transferred from the first
tissue collection container into a second collection container by a
closed system method. Such a closed system method may include, but
is not limited to, a mechanism such as a leur connector between the
first collection container and the second collection container, a
spike port, a needle, or combinations thereof. The closed system
method of transfer prevents the cell-enriched matrix that includes
a regenerative platform from being contaminated by any additional
pathogens external to the tissue sample and/or the tissue
collection containers, such as but not limited to bacteria,
viruses, and the like from entering into the tissue collection
containers or the cell-enriched matrix. The closed system decreases
the necessity for additional steps to be performed on the
cell-enriched matrix prior to the administration of the tissue
sample back into a subject as described above. As used herein, the
numeral notation of `first tissue sample container` and `second
tissue sample container` denotes the usage order of the containers.
The containers may be the same types of containers or different
types of containers, e.g., a vial or centrifuge tube.
[0111] The second tissue collection container may then be subjected
to at least one more acceleration and deceleration steps, as
described above. Each round of acceleration and deceleration may
occur until at a rate of at least about 10.times.g is obtained.
Alternatively, the rate of acceleration and deceleration may occur
at a rate ranging from about 10.times.g to independently about
400.times.g, or from about 20.times.g independently to about
40.times.g in another non-limiting embodiment. In one non-limiting
embodiment, the number of rounds per minute of acceleration and d
deceleration may range from about 1 round per minute to about one
round per five minutes, or alternatively at least about three
rounds per minute. In another non-limiting embodiment, the
cell-enriched matrix may be disaggregated after a number of rounds
of acceleration and deceleration, or alternatively at least two
rounds of acceleration and deceleration.
[0112] In some embodiments, one or more proteases (e.g., one or
more collagenases such as type I and/or type II collagenases, a
neutral protease such as thermolysin, trypsin, or mixtures thereof)
can be added to the second tissue container. One or more of the
proteases may be recombinantly produced. For example, one or more
collagenases can be added to the second tissue collection container
in an amount ranging from about 0.5 Wunsch units collagenase per ml
independently to about 4.0 Wunsch units collagenase per ml, or
alternatively from about 1.0 Wunsch units collagenase per ml
independently to about 3.0 Wunsch units collagenase per ml. The
intermittent rounds of acceleration followed by deceleration in the
presence of a protease may disaggregate the regenerative platform
of the cell-enriched matrix.
[0113] After the acceleration and deceleration steps, the
cell-enriched matrix may be filtered and washed to obtain a
regenerative platform that may be administered back into a subject
by implantation or injection as described above. For example, the
cell-enriched matrix can be filtered to obtain an injectable
regenerative platform, in which a few or no additional steps must
be performed for the regenerative platform to be injected into a
subject.
[0114] The invention will be further described with respect to the
following Example which is not meant to limit the invention, but
rather to further illustrate the various embodiments.
EXAMPLES
Example 1
[0115] Fresh canine omental adipose tissue was obtained from tissue
discarded after spay surgery. Tissue was minced with sterile
scissors and then equally divided (approximately 2 g/tube) into 50
mL sterile centrifuge tubes. Sterile lactated Ringer's containing a
blend of bacterial collagenases I and II together with dispase was
added and the tubes were then randomly assigned to incubation in a
shaking incubator (60 rpm) or a heated tissue processing apparatus
in a fixed rotor (TPA, 3 cycles per min of 1.times.g to 200.times.g
to 1.times.g). Temperature was maintained between 37-40.degree. C.
and incubation/processing was conducted for 30 min.
[0116] After processing, the dissociated tissue slurry was passed
through a 100 .mu.m filter and the cell fraction was recovered from
the filtrate by centrifugation at 400.times.g for 10 min in the
TPA. Cell fractions were plated in 25 cm.sup.2 tissue culture
flasks and grown for two days at 37.degree. C. in DMEM/20% (v/v)
fetal bovine serum (FBS) containing antibiotic and antimycotic.
After culturing for two days, adherent cells were counted using a
hemacytometer. FIG. 6 is a bar graph of the number of adherent
cells obtained after processing using the TPA and using the shaking
incubator. Processing the tissue with the TPA resulted in a 2.6
fold higher than when dissociatiion was performed in a shaking
incubator.
Example 2
[0117] Fresh human adipose lipoaspirate was obtained with patient
informed consent from a patient undergoing elective lipoplasty.
Tissue was drained using a sterile stainless steel strainer and
then equally divided (approximately 10 g/tube) into 50 ml sterile
centrifuge tubes with (see, FIGS. 3A, 3D, and 3E) or without
inserts fabricated from nylon mesh with 1 mm pore size. Sterile
lactated Ringer's containing a blend of bacterial collagenases I
and II together with dispase was added and the tubes then were
randomly assigned to incubation in a shaking incubator (60 rpm) or
a heated TPA in a swinging bucket rotor (3 cycles per min of
1.times.g to 200.times.g to 1.times.g). Temperature was maintained
between 37-40.degree. C. and incubation/processing was conducted
for 30 min.
[0118] After processing, the dissociated tissue slurry was passed
through a 100 .mu.m filter and the cell fraction was recovered from
the filtrate by centrifugation at 400.times.g for 10 min in the
TPA. Cell fractions were plated in 25 cm.sup.2 tissue culture
flasks and grown for two days in DMEM/20% (v/v) FBS containing
antibiotic and antimycotic. After culturing for two days, adherent
cells were counted using a hemacytometer. FIG. 7 is a bar graph of
the number of adherent cells obtained after processing using the
TPA and three different inserts, or after processing using the
shaking incubator. Results indicate that cell yield obtained by
processing in the TPA and using the cone insert (e.g., FIG. 3A) is
similar to cell yield obtained by processing in the incubator.
Example 3
[0119] A lipoaspirate sample was obtained from a human patient
undergoing elective lipoplasty. Lipoaspirate was transferred to a
plurality of tissue collection containers having a 20 cc volume.
Lipoaspirate contents of each of the tissue collection containers
were extruded five times through a micro-emulsifying needle. The
extruded lipoaspirate from each of the tissue collection containers
was then transferred to a separate tissue collection container. The
tissue collection containers were then placed into the cavities of
a removable rotating apparatus within an automated tissue
processing unit. The removable rotating apparatus allowed the
tissue collection containers to maintain a horizontal position,
while the automated tissue processing unit applied acceleration and
centrifugal force to the contents of the tissue collection
containers. The centrifugal force was applied for 30 minutes at a
rate of about 400.times.g, about 700.times.g, about 1200.times.g,
or about 2000.times.g for 30 minutes. The contents of two tissue
collection containers were used for each rate of centrifugal
force.
[0120] After the centrifugation, a cell-enriched matrix having a
regenerative platform was separated from the remainder of the
tissue sample within each tissue sample collection container. The
volume of the layer of oil, a lipoaspirate fraction, and an aqueous
fraction was removed and measured for each sample. The lipoaspirate
layer was placed into a separate 50 cc conical centrifuge tube.
[0121] As a control sample, approximately 5 g of unprocessed, i.e.,
neither extruded nor centrifuged, lipoaspirate was transferred to
each of 2 tissue collection containers. The containers were weighed
and the weight of the transferred lipoaspirate was recorded.
Ringer's lactate that includes a collagenase enzyme and a dispase
enzyme was added to the unprocessed lipoaspirate in an amount of 5
mL to each tissue collection container. The tissue collection
containers were then placed into a shaking incubator at about
37.degree. C., and about 60 rpm for about 30 min to disaggregate
the unprocessed lipoaspirate. The disaggregated tissue sample was
then passed through a 100 .mu.m steriflip filter. The filtered
tissue was then centrifuged at a rate of about 600.times.g for
about 10 minutes to recover regenerative cells. The recovered cells
were placed in culture in minimum essential medium (MEM) with 20%
(v/v) fetal bovine serum for 24 hours. The adherent cells were
counted by a hemacytometer.
[0122] As illustrated in FIG. 10, extruding the tissue sample prior
to centrifugation yields a greater number of cells per gram of
tissue. FIG. 11 illustrates that centrifugation at 1200.times.g has
an additive effect in terms of increasing the cell concentration
within the cell-enriched matrix regardless of whether the tissue
was extruded prior to centrifugation.
[0123] FIG. 12 illustrates that a longer amount of time for
centrifugation of the tissue sample yields a higher number of cell
per gram of cell-enriched matrix. FIG. 13 illustrates that a
1200.times.g rate of acceleration yields a higher concentration of
cells per cell-enriched matrix when compared to a 400.times.g rate
of acceleration or no centrifugation at all.
Example 4
[0124] Fresh human lipoaspirate was divided into two aliquots.
Aliquot 1 ("control method") was processed using conditions similar
to those commonly employed in cosmetic surgery to prepare
lipoaspirate derived fat graft. The lipoaspirate was centrifuged
for 2 minutes at 200.times.g. Aliquot 2 ("cell-enriched matrix"
(CEM) method) was processed by first extruding the lipoaspirate
across a luer coupling between two syringes 5 times, and then
centrifuging the extruded lipoaspirate for 30 minutes at
1200.times.g. After centrifugation, both methods resulted in
fractionation into an upper oil layer, a middle tissue layer, and a
lower aqueous layer. The middle tissue layer fraction of each
aliquot was collected. A portion of the collected tissue layer
fraction from each method was loaded into individual 1 cc syringes
and administered subcutaneously into the nuchal area of female
immunodeficient NU/NU mice (n=3 mice/preparation).
[0125] An additional portion of the tissue layer fraction from each
method was processed at 37.degree. C. with a blend of collagenases
I and II and dispase, filtered through a 100 .mu.m filter, and then
centrifuged at 600.times.g to obtain the regenerative cells. Number
of viable regenerative cells in the fresh cell preparations and
number of plastic adherent cells in culture at 24 h were
determined. At 1 month post-implantation, mice were sacrificed and
grafts were evaluated.
[0126] Processing by the CEM method resulted in a 2.2 fold higher
concentration of viable cells in the fresh preparation and a 5.5
fold higher concentration of plastic adherent cells compared to the
control method. See Table 1. This cell enrichment translated to a
higher viability of the graft at 1 month as evidenced by
vascularization and absence of oil pockets in mice injected with
the cell preparations obtained using the CEM method.
TABLE-US-00001 TABLE 1 Method Viable cells/g tissue Adherent
cells/g tissue Control 2.82 .times. 10.sup.5 5.35 .times. 10.sup.4
CEM 6.16 .times. 10.sup.5 2.94 .times. 10.sup.5
OTHER EMBODIMENTS
[0127] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
* * * * *